Characterization of NDM-5-Producing Escherichia coli Strains Isolated from Pediatric Patients with Bloodstream Infections in a Chinese Hospital

Escherichia coli (E. coli) bloodstream infections (BSIs) are among the most predominant causes of death in infants and children worldwide. NDM-5 (New Delhi Metallo-lactamase-5) is responsible for one of the main mechanisms of carbapenem resistance in E. coli. To analyze the phenotypic and genomic characteristics of NDM-5-producing E. coli from bloodstream infections (BSIs), a total of 114 E. coli strains was collected from a children’s hospital in Jiangsu province, China. Eight blaNDM-5-carrying E. coli strains were identified which were all carbapenem-resistant and carried diverse antimicrobial resistance genes apart from blaNDM-5. They belonged to six distinct sequence types (STs) and serotypes including one each for ST38/O7:H8, ST58/O?:H37, ST131/O25:H4, ST156/O11:H25 and ST361/O9:H30 and three strains are originating from a single clone belonging to ST410/O?:H9. Apart from blaNDM-5, the E. coli strains isolated from BSIs also carried other β-lactamase genes, including blaCMY-2 (n = 4), blaCTX-M-14 (n = 2), blaCTX-M-15 (n = 3), blaCTX-M-65 (n = 1), blaOXA-1 (n = 4) and blaTEM-1B (n = 5). The blaNDM-5 genes were located on three different types of plasmids, which were IncFII/I1 (n = 1), IncX3 (n = 4) and IncFIA/FIB/FII/Q1 (n = 3). The former two types were conjugatively transferable at frequencies of 10−3 and 10−6, respectively. The dissemination of NDM-producing strains, which exhibit resistance to the last-line antibiotics, carbapenems, may increase the muti-antimicrobial resistance burden among E. coli BSIs and further threaten public health.


Introduction
Bloodstream infections (BSIs) are considered among the most serious nosocomial pathologies with limited choices of effective therapeutic options. BSIs are frequently associated with high morbidity and mortality in hospitalized patients, which increases treatment costs, patients' burden and diagnostic uncertainties [1]. According to the published data, 48.9 million incident cases have taken place worldwide, and 11.0 million deaths were associated with BSIs, which represented 19.7% of all global deaths [2]. Of note, BSIs remain a major problem for neonates and young children [3,4], because the diagnosis of BSIs in children is relatively difficult as they could manifest as serious infections in other sites of over many countries [38]. NDM-type carbapenemases mediate resistance to most β-lactams antibiotic except aztreonam [39]. NDM variants are increasingly becoming one of the main effectors of carbapenem resistance among E. coli isolates in clinical settings in China [40]. NDM-positive E. coli isolated from BSIs were reported from different countries across the world, including India [41], China [42], Sweden [43], Cuba [44] and several Latin American countries [45]. Despite their prevalence being relatively low, such strains could cause considerable mortality and pose great challenges to the treatment of bloodstream infections [46].
At the time of writing, a total of 44 NDM variants, namely, NDM-1 to NDM-44, were deposited in the database (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#gene_ family: (bla NDM ) (accessed on 1 November 2022)). Among these, NDM-5 was firstly detected in E. coli isolated from the throat of a patient with a history of travel to India in 2011 [47] and subsequently reported in different countries including India [48], South Korea [49], Australia [50], America [51] and Egypt [52]. NDM-5 was also reported to be the dominant NDM variant produced by E. coli strains from different hospitals in Jiangsu province, China [53,54].
Yet, the phenotypic and genetic characteristics of the clinical NDM-5-producing E. coli strains remain largely unknown. To fill this gap, we collected a total of eight NDM-5-producing E. coli strains isolated from between years 2016 and 2020 from BSIs in a children's hospital in Jiangsu, China. The clinical information of the studied strains was also collected. The antimicrobial resistance phenotypes and the genetic characteristics of all strains were comprehensively studied. In this study, we conducted a bioinformatics analysis to determine the molecular characteristics of NDM-5-positive E. coli carried by pediatric patients and assessed the transmission ability of the plasmid harboring bla NDM-5 . Our findings shall increase the understanding of how to control of untreatable BSIs caused by carbapenem-resistant E. coli.

Bacteria Isolates and Clinical Data Collection
A total of 114 non-duplicated E. coli strains isolated from blood specimens from 114 patients from the Children's Hospital of Soochow University (Suzhou, China) were collected during the period from September 2016 to October 2020. The total numbers of E. coli isolates during these periods were 1 in 2016 and 2019, respectively, 3 in 2017 and 3 in 2020. The strains were isolated in accordance with standard procedures. Briefly, the blood samples were first cultured with the BD BACTEC TM FX Blood Culture System (BD Diagnostics, Sparks, MD, USA). The positive cultures were sub-cultured on blood agar and in Mueller-Hinton broth at 37 • C for 16-24 h to obtain the E. coli isolates. All E. coli isolates from patients meeting the inclusion criteria of the study were stored in the laboratory at -80 • C in cryovials containing 20% glycerol and the nutrient broth for further analysis. The clinical information of the patients from whom the bla NDM-5 -positive E. coli isolates were isolated was obtained from the clinicians. The relevant clinical information and the origin of the strains are presented in Table 1.

Conjugation Assay
A conjugation assay was performed using the filter-mating method [57]. Briefly, each bla NDM-5 -carrying E. coli strain was used as the donor, and rifampicin-resistant E. coli EC600 was used as the recipient to test the transferability of the carbapenem resistance gene, bla NDM-5 . The donor and the recipient were mixed in the ratio of 1:1 and cultured overnight on a nitrocellulose filter (HA type; pore size, 0.22 µm; Millipore Corp, Billerica, MA, USA) placed on LB agar. The filters were removed from the plates and placed in 1.0 mL of phosphate buffer. The cells were removed from the filter with a Vortex mixer. Dilutions were made, and all transconjugants were selected on LB agar plates supplemented with 1 µg/mL of meropenem and 600 µg/mL of rifampicin. The transconjugants were confirmed by PCR targeting the bla NDM-5 gene and by antimicrobial susceptibility testing. The conjugation frequencies were estimated by dividing the number of colonyforming units (CFUs) of the transconjugants by the number of CFUs of the recipient strains E. coli EC600.

DNA Extraction
The genomic DNA of each bla NDM-5 -carrying E. coli was extracted from overnight cultures on BHI using a Genomic DNA Extraction Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. The purity and concentrations of DNA were measured by a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Whole-Genome Sequencing and Bioinformatics Analysis
The extracted genomic DNA was subjected to whole-genome sequencing using the HiSeq platform (Illumina, San Diego, CA, USA). Trimmomatic was used to trim the raw reads in order to remove the adaptors and low-quality sequences [58]. De novo genome assembly was conducted with SPAdes v3.15.5 [59]. The draft genome sequences were annotated using RAST [60]. Acquired antimicrobial resistance genes (ARGs) were identified with ResFinder 4.1 [61]. Insertion sequences (ISs) were identified with the online software ISfinder [62]. Plasmid replicon types were identified with PlasmidFinder [63]. The comparison of the plasmids was performed using BLASTn, and the results were analyzed with the R tool [64]. The O:H serotype was identified with ECTyper v0.8.1 [65]. MLST v2.11 was used to determine the variety of STs [66]. Variant calling and core genome alignment of the strains E3, E4 and E5 was conducted using Snippy 3.1 [67]. Pairwise SNP was calculated using snp-dists 0.8.2 [68]. The harvest suite v.1.2 was used to remove recombination sequences and conduct the phylogenetic analysis using the assembled genome sequences as the input [69]. The generated phylogenetic tree was visualized and modified with iTOL v6 [70]. A heatmap of antimicrobial resistance genes was obtained using TBtools [71]. Plasmid alignment was visualized with BRIG [72].

Ethical Statements
For the study was obtained through the Children's Hospital of Soochow University (Suzhou, China).

Clinical Information and Overview of the NDM-5-Producing E. coli Strains
Among the 114 E. coli strains from BSIs collected from 2016 to 2020 in a children's hospital in China, 8 (7.02%) strains designated E1-E8 carried bla NDM-5 . The age of the eight patients ranged from 9 days to 13 years. Three (37.5%) were male patients, and five (62.5%) were female patients. The patients suffered from different diseases including acute myeloid leukemia, acute lymphoblastic leukemia, neonatal sepsis, congenital atresia of the biliary tract, etc., and were admitted to different departments in the hospital including hematology (n = 4), neonatology (n = 3) and gastroenterology (n = 1). The patients were treated with different antibiotics such as meropenem, tigecycline and amikacin. Seven patients recovered, and one patient was discharged automatically (Table 1).

Antimicrobial Resistance Profiles of the NDM-5-Producing E. coli Strains
The minimum inhibitory concentrations of commonly used antibiotics for the NDM-5producing E. coli strains are listed in Table 2. All these strains exhibited multidrug resistance phenotypes. All eight strains were resistant to ampicillin, imipenem, cefepime, ceftriaxone, ceftazidime, cefotetan, ampicillin-sulbactam, piperacillin-tazobactam and trimethoprimsulfamethoxazole. The proportion of E. coli strains that were non-susceptible to aztreonam, gentamycin and tobramycin were 62.5%, 50% and 75%, respectively. In addition, 87.5% of the eight strains were resistant to ciprofloxacin, and the remaining 12.5% strains exhibited intermediate resistance to ciprofloxacin. We found that 62.5% and 37.5% of the strains were resistant and intermediately resistant to levofloxacin, respectively.

Transferability and Genetic Analysis of blaNDM-5-Carrying Plasmids
To further investigate the role of NDM-5-producing E. coli strains in bacterial dissemination, we analyzed the drafts of the whole-genome sequences of all strains and found that the blaNDM-5 genes were located in contigs ranging from 3 Kb to 43 Kb. The blaNDM-5 gene in strain E2 was located on a 43,563 bp contig, and the blaNDM-5 genes in other strains were all located on contigs < 9 Kb. The blaNDM-5-carrying contigs were generally too short to accurately resolve the genetic context of blaNDM-5. The blaNDM-5-bearing contigs in these Heatmap of antimicrobial resistance genes carried by E. coli isolates in this study. The horizontal axis represents the antimicrobial resistance genes, and the vertical axis represents the strain IDs. The red boxes represent the presence of the corresponding items among the sequenced isolates, and the white boxes represent their absence. The gradient identity bar indicates the percentage similarity of related genes. The similarity tree was constructed using agglomerative hierarchical clustering, with the degree of similarity between different clusters calculated by the average linkage method and the degree of similarity of different isolates (calculated using the Spearman's rank correlation coefficient).The classes of antibiotics to which the genes confer resistance included aminoglycosides: strA, strB, aac(3)-IId, aac(6)-Ib-cr, aadA2, aadA5, aph3-Ia, aph(4)-Ia and aac (3)

Discussion
Carbapenems are used as the last-resort antibiotics to treat Gram-negative bacterial infections. The emergence of carbapenem-resistant organisms is considered one of the most urgent public health concerns [73]. The acquisition of carbapenemase genes by Gram-negative strains is one of the major mechanisms of carbapenem resistance, and the horizontal gene transfer of carbapenemase genes located on mobile genetic elements, including transposons and plasmids, is causing a widespread dissemination of CRE [37]. E. coli is among the most frequently isolated CRE worldwide which could colonize both elderly patients (aged >60 years) and the pediatric population [74,75]. Carbapenem-resistant E. coli isolated from blood samples has been reported across the world [76]. Carbapenemresistant E. coli strains carrying different blaNDM gene variants were found in North America, Africa, Asia and Europe [77]. A study from hospitals in Chongqing, China, on strains from BSIs suggested that the infection ratio of E. coli in children was significantly higher than in adults, and most of the E. coli strains from BSIs in children expressed blaNDM, particularly blaNDM-5 [78]. Furthermore, the spread of ESBL-producing E. coli is one of the most important driving factors of the abuse of carbapenems, which indirectly exacerbates the selective pressure of carbapenemase producers [79]. It is well known that CTX-M-15 and CTX-M-14 are the most common CTX-Ms worldwide, often reported in South-East Asia [27]. Our study also demonstrated that carbapenemase-producing E. coli are often multidrug resistant and usually express other antibiotic resistance genes.

Discussion
Carbapenems are used as the last-resort antibiotics to treat Gram-negative bacterial infections. The emergence of carbapenem-resistant organisms is considered one of the most urgent public health concerns [73]. The acquisition of carbapenemase genes by Gram-negative strains is one of the major mechanisms of carbapenem resistance, and the horizontal gene transfer of carbapenemase genes located on mobile genetic elements, including transposons and plasmids, is causing a widespread dissemination of CRE [37]. E. coli is among the most frequently isolated CRE worldwide which could colonize both elderly patients (aged >60 years) and the pediatric population [74,75]. Carbapenem-resistant E. coli isolated from blood samples has been reported across the world [76]. Carbapenemresistant E. coli strains carrying different bla NDM gene variants were found in North America, Africa, Asia and Europe [77]. A study from hospitals in Chongqing, China, on strains from BSIs suggested that the infection ratio of E. coli in children was significantly higher than in adults, and most of the E. coli strains from BSIs in children expressed bla NDM , particularly bla NDM-5 [78]. Furthermore, the spread of ESBL-producing E. coli is one of the most important driving factors of the abuse of carbapenems, which indirectly exacerbates the selective pressure of carbapenemase producers [79]. It is well known that CTX-M-15 and CTX-M-14 are the most common CTX-Ms worldwide, often reported in South-East Asia [27]. Our study also demonstrated that carbapenemase-producing E. coli are often multidrug resistant and usually express other antibiotic resistance genes.
The eight NDM-5-producing strains belonged to six different sequence types including ST410, ST38, ST58, ST131, ST361 and ST156, all of which have been reported to be associated with multidrug resistance phenotypes. Among the sequence types in this study, ST410, ST38, ST361 and ST156 E. coli were identified as NDM-5-producers in previous studies [53,[82][83][84]. ST131 and ST58 E. coli have been reported to be NDM-1 producers [85,86]. Previous epidemiological studies demonstrated that the dominant E. coli sequence type associated with BSIs in China was ST131, followed by ST69 and ST38 [87,88]. ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide and is commonly reported to produce extended-spectrum β-lactamases [32]. Previous studies reported that CTX-M-14 and CTX-M-15 were the most common ESBLs in E. coli ST131 isolates [32], which is in line with the results of our study. Two serotypes of ST131, O16:H5 and O25b:H4 have been identified [34]. In 2008, the serogroup O25b and the sequence type ST131were detected in many countries from analyses of the population biology of ESBLproducing E. coli [89]. The strain E6 in this study belongs to O25b:H4, ST131. Due to the limited number of strains included in this study, we were unable to compare the prevalence of different sequence types that we calculated with that reported in the literature.
Three strains, belonging to a single clone with the sequence type ST410 and the serotype O?:H9, were collected in this study. ST410 has been reported worldwide as an extraintestinal pathogen associated with resistance to different classes of antibiotics such as fluoroquinolones, polymyxins, third-generation cephalosporins and carbapenems [33,90]. ST410 was further classified into several clades, each of which is associated with a different serotype, including clades A and B (serotype O8:H21), clade C (O8:H9), and clade D and E (O?:H9) [35]. We found that the strains E3, E4 and E5 recovered from bloodstream infection belonged to the serotype clades D and E, which has not been published before. Strains belonging to the clades D and E could be recovered from human and animal samples, posing threat to both populations [35]. The clone ST410 E. coli undergoes constant evolution, with its resistome changing with time. Around 2014, a clade of ST410 acquired the carbapenemase gene, bla NDM-5 , on a conserved IncFII plasmid; this could be the ancestral source of the bla NDM-5 -carrying plasmids in the ST410 strains E3, E4 and E5 analyzed in this study [33]. A previous study reported the isolation of an ST410 E. coli strain a carrying bla NDM-5 -encoding IncX3 plasmid from a 59-year-old patient with BSI in China, yet the ST410 E. coli carrying a bla NDM-5 -encoding IncFII plasmid isolated from blood samples of patients with BSIs was not reported before this study [91]. In addition, we reported the first ST410 clone which co-harbored five β-lactamase genes, including bla NDM-5 , bla CMY-2 , bla CTX-M-15 , bla OXA-1 and bla TEM-1B . Considering that the ST410 clone isolated in this study carries a cassette of important resistance genes, has the potential of transmission between patients and could cause BSIs, it should be monitored closely in the future.
Plasmids play a pivotal role in the dissemination of antimicrobial resistance genes. bla NDM-5 has been reported to be closely associated with different types of plasmids such as IncX3, IncFII and IncHI2 [91][92][93]. Among these, the IncF plasmid, with a low copy number, has a narrow host range (Enterobacteriaceae) and has greatly improved the fitness cost through its antimicrobial resistance determinants by horizontal gene transfer [94]. This is the most common plasmid type in the ST131 clone and is widely distributed in clinically Enterobacteriaceae. The IncX3 plasmid containing bla NDM-5 has showed a varied geographical distribution in China, as it has been isolated in Jiangsu province [95], Shanghai [96] and Zhejiang province [97]. The bla NDM-5 genes in the strains E1−E8 were all plasmid-borne. Three different types of bla NDM-5 -carrying plasmids were identified among the strains, including the IncFII/I1 pE−T654−NDM-5−like plasmids, the IncX3 pL65−9−like plasmids and the IncFIA/FIB/FII/Q1 pC405−NDM5−like plasmids. The former two types carry conjugation-associated genes and are conjugatively transferable at different frequencies. The pC405−NDM5−like plasmids are non-self-transmissible. The reason that bla NDM-5 in the strains E3, E4 and E5 are non-conjugative transferable could be explained by the fact that the IncFIA/FIB/FII/Q1 pC405−NDM5−like plasmid does not carry conjugation-associated genes. The fact that no transconjugants were detected for strain E2 could be explained by a low conjugation frequency (~10 −6 ) of the IncX3 plasmids. The transferability of bla NDM-5 -carrying plasmids may promote the rapid dissemination of resistance-encoding elements among Gram-negative bacterial pathogens. Controlled measures such as optimizing antibiotic usage and the development of new antibiotics or antibiotic adjuvants are urgently needed to prevent the emergence and transmission of resistant bacteria such as carbapenem-resistant E. coli.

Conclusions
In summary, this study collected 8 bla NDM-5 -carrying E. coli strains from a total of 114 E. coli strains from bloodstream infections in a children's hospital in Jiangsu province, China. These strains were carbapenem-resistant and carried diverse antimicrobial resistance genes apart from bla NDM-5 . The eight strains belonged to six distinct STs and serotypes including one each for ST38/O7:H8, ST58/O?:H37, ST131/O25:H4, ST156/O11:H25 and ST361/O9:H30, and three strains are originating from a single clone belonging to ST410/ O?:H9. Five β-lactamase genes were identified in the ST410 E. coli strains, i.e., bla NDM-5 , bla CMY-2 , bla CTX-M-15 , bla OXA-1 and bla TEM-1B . The bla NDM-5 gene was located on three different types of plasmids, which were IncFII/I1 (n = 1), IncX3 (n = 4) and IncFIA/FIB/FII/Q1 (n = 3). The former two types were conjugatively transferable at frequencies of 10 −3 and 10 −6 , respectively. The results of this study could increase our understanding of carbapenem-resistant E. coli from BSIs. Effective strategies such as the development of new antibiotics or antibiotic adjuvants and the optimization of antibiotic usage are needed for the control of untreatable BSIs caused by carbapenem-resistant E. coli.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by an independent ethics committee and the Institutional Review Board of Children's hospital of Soochow University.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The assembled genome sequences of all E. coli strains from bloodstream infections in this study were deposited in the NBCI database under BioProject accession number PRJNA890582.

Conflicts of Interest:
The authors declare no conflict of interest.